Specimens

Animals and sample preparation: Sprague Dawley rats (female and male, 250-300 g; Taconic Farms, Germantown, NY) were maintained on a 12:12 light:dark (L:D) cycle. Animal use and care protocols were approved by local ethical review and were in accordance with National Institutes of Health guidelines.

Single-cell Preparation

Cell preparation: Two pools of glands were composed of tissue obtained from two males and two females that had been CO2-anesthetized at mid-day, 6 hours after lights on Zeitgeber time (ZT) 0600. Pineal glands were rapidly removed and placed into DMEM culture medium (4°C). Single cells were obtained using the Papain Dissociation System (Worthington; Lakewood, NJ) according to the manufacturer’s instructions, modified for pineal cells as described in Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression.

Single-cell RNA-Seq library preparation and sequencing

Library preparation and sequencing: Cells were captured using a Chromium Single Cell Processor (10X Genomics; Pleasanton, CA) and single-cell cDNA libraries were prepared using a Chromium Single Cell 3′ Reagent Kit v2 following the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq2500 (Illumina; San Diego, CA), generating 98 bp of sequence adjacent to the polyA tail. Approximately 3,000 cells per sample were recovered, with approximately 50k reads per cell and 2,700 to 3,000 genes per cell detected on average.

Bioinformatics methods

See bioinformatics methods for details.

References

1. Mays, JC, Kelly MC, Coon SL, Holtzclaw L, Rath MF, Kelley MW, Klein DC. Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression PLoS ONE 13(10):e0205883. doi: 10.1371/journal.pone.0205883.